Mitochondria (mt) play key roles in cellular energy production and cell death. Beta cell function is tightly linked to mitochondria; as both insulin synthesis and glucose stimulated insulin secretion require mitochondrial ATP production. In this context, reports of mitochondrial DNA (mtDNA) mutations associated with diabetes (T2D) pedigrees in humans account for up to 1% of human T2D. However, mutations in mtDNA are not commonly associated with autoimmune Type 1 diabetes (T1D), although a C to A transversion resulting in a leucine to methonine substitution in the mt-ND2 gene has been associated with protection from T1D in both an at risk human population and in crosses of the T1D-prone NOD with T1D-resistant ALR mice. The goal of this application is to understand how this single amino acid change modifies resistance to T1D. Genetic analysis of T1D susceptibility has focused attention on candidate genes controlling aberrant immune cell function with little focus on genes that may contribute susceptibility or resistance at the cell level. Pancreatic islets from the ALR mouse strain maintain an unusual genetic resistance to functional impairment and killing by autoimmune effectors. Our results have linked some of the ALR-derived T1D resistance to mt- Nd2a. While this allele does not prevent the development of spontaneous T1D when introgressed into the NOD genetic background, the ALR-derived genetic variant does in fact protect beta cells from destruction by cytotoxic T lymphocytes as well as combined cytokines. We have determined that this resistance stems from the inability of pro-apoptotic stress to induce mt reactive oxygen species. Our goal is to understand the role this gene plays in cell resistance to autoimmune killing. In specific aim 1 we will determine the mechanism by which mt-Nd2a encoding cells and primary islets resist destruction mediated by members of the Tumor Necrosis Factor Receptor family, TNFR1 and Fas. Aim 2 will extend the studies to determine the mechanisms utilized by mt-Nd2a encoding islet cells to avert lysis by CTL. Specific aim 3 will use human islets to confirm mechanisms of resistance to cell destruction as well as cybrid human cells to test if the human alleles of mt-ND2 alter the characteristics of human cell death. The studies, as proposed, will provide meaningful data on both the early apoptotic signals that result in human beta cell death as well as insights into how ND2a protects human beta cells. PUBLIC HEALTH RELEVANCE: The genetics of susceptibility versus resistance to autoimmune T1D has primarily focused attention on candidate genes controlling function of the immune system rather than on the target beta cell. The assumption has been that insulin producing beta cells express a common repertoire of target antigens, yet play no other role in T1D pathogenesis than the corpse. In contrast to this commonly held view, our work has clearly shown that genetics play an important role in resistance of the target tissue to the autoimmune destruction leading to Type 1 Diabetes. The current proposal exploits a genetic difference that provides resistance to autoimmune- mediated destruction in both human and mouse. The objectives of this proposal are to determine the mechanism this gene variant employs to prevent beta cell death and also to identify the early pro-death signals that occur in beta cells exposed to autoimmune effectors. The long-term goal of this work is to identify how the mt-ND2a allele provides protection against T1D in human populations. Protective factors human pancreatic islets can employ to ward off autoimmune-mediated destruction mechanisms have important ramifications for transplantation, stem cell engineering, and future genetic as well as pharmacological diabetes preventative therapies.